Formins promote actin assembly in eukaryotes, strongly stimulating both filament nucleation and elongation. It remains unclear how their potent activities are controlled in vivo with the spatial and temporal precision required to assemble actin networks with particular architectures and dynamics. In the budding yeast S. cerevisiae, I will combine genetic and biochemical approaches to study how the formin Bnr1 is controlled temporally. From the bud neck, Bnr1 assembles dynamic actin cables that extend into the mother cell. These serve as tracks for the polarized traffic of secretory vesicles by Myosin V (Myo2). We have shown that the kinesin-like protein Smy1 binds and inhibits Bnr1 actin assembly in vitro. SMY1 deletion in vivo causes striking defects in actin cables, and we hypothesize that these stem from a loss of Bnr1 regulation in the absence of Smy1.Specific Aim 1: Define the biochemical mechanism of Smy1 inhibition of Bnr1. In our model, Smy1 binds Bnr1 at the ends of actin filaments and slows the rate of elongation without displacing Bnr1 from the filament. To test this, I will use in vitro multi-color TIRF microscopy to visualize the dynamics of Bnr1 molecules on filaments in the presence and absence of Smy1, while measuring filament elongation rates. Further, I will simultaneously image Smy1 and Bnr1 (differentially labeled) to quantify the dynamics of their interactions with each other and with filaments. Finally, I will challenge Bnr1/Smy1-capped filament ends with capping protein, which is predicted to compete with Bnr1, to reveal whether Smy1 affects Bnr1 dissociation from filaments under competitive conditions.Specific Aim 2: Dissect the in vivo functions and dynamics of Smy1. We have shown that Myo2 rapidly transports Smy1 on actin cables. Further, the actin cables in smy1-null cells are abnormally long and change direction multiple times at sharp angles. Our model is that Smy1 inhibition of Bnr1 controls actin cable length. However, Smy1 also enhances Myo2 motor activity, raising the possibility that the cable defects in smy1-null cells instead arise from loss of Myo2 regulation. I will test these competing models by generating alleles of smy1 that disrupt association with Bnr1 but not Myo2, and analyze the alleles for effects on actin cable assembly and secretory vesicle movement. This will test the physiological importance of Smy1-Bnr1 interactions, and whether effects of Smy1 on Bnr1 and Myo2 are coordinated in vivo.
|Program type||Predoctoral Fellowship|
|Effective start/end date||07/01/2011 → 06/30/2013|